Anthrax is caused by the spore-forming bacterium Bacillus anthracis. The ability to deliver the spores in an aerosol and the high mortality rate of inhalational anthrax have led to the use of the spores as a biological weapon. Antibiotic treatment of inhalational anthrax can be ineffective late in the infection because high levels of toxin in the blood cause death. Our research proposal is focused on the development of inhibitors of anthrax toxin, a combination of three proteins secreted by the bacterium. Protective antigen (PA) binds a receptor and is cleaved by a protease, allowing the heptamerization of the cell-associated PA63 fragment. Heptamerization allows binding of the enzymatic toxin components, edema factor (EF) and lethal factor (LF), and triggers endocytosis of these complexes. The acidic environment of the endosome leads to the translocation of the enzymatic proteins to the cytosol where they exert their toxic effects. EF is an adenylate cyclase that impairs the innate immune response by a variety of mechanisms. LF is a protease that causes lysis of macrophages, which results in shock-like symptoms and death. The recent identification of the anthrax toxin receptor (ATR) will facilitate the development of molecules that inhibit anthrax toxin action. We will use two approaches to isolate inhibitors of the PA-ATR interaction and test these inhibitors for the ability to block the intoxication process in vitro. In the first approach, we will select a peptide that binds ATR and then attach multiple copies of this peptide to a polymeric backbone. The resulting polyvalent compound is predicted to bind ATR with higher affinity than the peptide alone and prevent binding of PA to cells. The second approach is based on our previous observation that a soluble fragment of ATR can protect cells from toxin in vitro. We hypothesize that a mutant fragment that binds PA with higher than wild-type affinity will be a more effective inhibitor. We will isolate this mutant through sequential rounds of random mutagenesis, selection, and recombination. These compounds may extend the time during which a case of anthrax can be treated successfully.